Cell Ig Research Articles

FGL2 promotes tumor progression via inducing TIGIT expression on T cells in tumor microenvironment of glioma

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor with a median survival of only 14.6 months. Our previous work shows that FGL2 promotes glioma tumor growth and the expression levels of FGL2 positively correlated with glioma grade in patients. T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here we found that there is a statistically significant positive correlation between FGL2 and TIGIT mRNA expression level in both low grade glioma database (TCGA, provisional) and glioblastoma multiforme database (TCGA, provisional). Future, FGL2 and TIGIT are coordinately expressed by human tumor-infiltrating lymphocytes of glioma as detected by flow cytometry. In addition, TIGIT expression on T cells which suppresses their function, can be induced by initial T cell activation or FGL2 in vitro. Induced TIGIT expression upon initial T cell activation is suppressed in FGL2KO T cells which has higher tumor-killing ability than WT T cells. Therefore, our results show that FGL2 induces TIGIT expression in brain-infiltrated lymphocytes which suppresses anti-tumor immune response in glioma. By this mechanism, FGL2 promotes tumor progression of glioma.

Stromal cells from inflamed lymph nodes modulate B cell surface CD23 expression

Abstract CD23 is a trans-membrane protein belonging to the C-type lectin family, widely expressed in the immune system. Originally identified as the low-affinity receptor for IgE, CD23 was later shown to also bind CD21, MHCII and various integrins, and to have pleiotropic roles, including modulation of cell proliferation, differentiation, Ig and cytokine secretion. Expression of membrane CD23 (mCD23) is regulated at least in part by its shedding from the cell surface via activity of metalloproteinases, the most important of which is ADAM10. Interestingly, secreted CD23 (sCD23) isoforms can exert immunomodulatory functions as soluble factors. B cells upregulate mCD23 and serum sCD23 levels have been found to be elevated in inflammatory and autoimmune conditions, including rheumatoid arthritis, and in B cell chronic lymphocytic leukemia. However, the mechanisms responsible for CD23 modulation on B cells are still unclear. Here we show that mCD23 is upregulated on mouse and human B cells found in inflamed lymphoid tissues. Furthermore, in vitro experiments demonstrate that B cell mCD23 upregulation is strictly dependent on soluble signals provided by stromal cells from inflamed, but not non-inflamed lymph nodes, and can be reversed by ADAM10-specific inhibitors. We propose therefore that the balance between B cell mCD23 and sCD23 expression in inflammatory conditions is modulated by microenvironmental signals provided by stromal cells within lymphoid tissues.

TIGIT Enhances Antigen-Specific Th2 Recall Responses and Allergic Disease.

T cell Ig and ITIM domain receptor (TIGIT), expressed on T, NK, and regulatory T cells, is known as an inhibitory molecule that limits autoimmunity, antiviral and antitumor immunity. In this report, we demonstrate that TIGIT enhances Th2 immunity. TIGIT expression was upregulated in activated Th2 cells from mice with experimental allergic disease and in Th2 polarization cultures. In addition, its high-affinity ligand CD155 was upregulated in mediastinal lymph node dendritic cells from allergic mice. In an in vitro setting, we observed that Tigit expression in Th2 cells and its interaction with CD155 expressed in dendritic cells were important during the development of Th2 responses. In addition, blockade of TIGIT inhibited Th2, but had no effect on either Th1 or Th17 polarization. In vivo blockade of TIGIT suppressed hallmarks of allergic airway disease, such as lung eosinophilia, goblet cell hyperplasia, Ag-specific Th2 responses, and IgE production, and reduced numbers of T follicular helper and effector Th2 cells. Thus, TIGIT is critical for Th2 immunity and can be used as a therapeutic target, especially in light of recent findings showing TIGIT locus hypomethylation in T cells from pediatric patients with allergic asthma.

High-Resolution Longitudinal Study of HIV-1 Env Vaccine-Elicited B Cell Responses to the Virus Primary Receptor Binding Site Reveals Affinity Maturation and Clonal Persistence.

Because of the genetic variability of the HIV-1 envelope glycoproteins (Env), the elicitation of neutralizing Abs to conserved neutralization determinants including the primary receptor binding site, CD4 binding site (CD4bs), is a major focus of vaccine development. To gain insight into the evolution of Env-elicited Ab responses, we used single B cell analysis to interrogate the memory B cell Ig repertoires from two rhesus macaques after five serial immunizations with Env/adjuvant. We observed that the CD4bs-specific repertoire displayed unique features in the third CDR of Ig H chains with minor alterations along the immunization course. Progressive affinity maturation occurred as evidenced by elevated levels of somatic hypermutation (SHM) in Ab sequences isolated at the late immunization time point compared with the early time point. Abs with higher SHM were associated with increased binding affinity and virus neutralization capacity. Moreover, a notable portion of the CD4bs-specific repertoire was maintained between early and late immunization time points, suggesting that persistent clonal lineages were induced by Env vaccination. Furthermore, we found that the predominant persistent CD4bs-specific clonal lineages had larger population sizes and higher affinities than that from the rest of the repertoires, underscoring the critical role of Ag affinity selection in Ab maturation and clonal expansion. Genetic and functional analyses revealed that the accumulation of SHM in both framework regions and CDRs contributed to the clonal affinity and antigenicity evolution. Our longitudinal study provides high-resolution understanding of the dynamically evolving CD4bs-specific B cell response after Env immunization in primates.

Local induction of IgT responses to pathogens and microbiota in the gill of rainbow trout

Gas exchange structures are critical for acquiring oxygen, but they also represent portals for pathogen entry. Local mucosal immunoglobulin (Ig) responses against pathogens in specialized respiratory organs have only been described in tetrapods. We have previously shown that IgT is an Ig specialized in gut and skin mucosal immunity. Thus, we tested the hypothesis that IgT might play a pivotal role in mucosal immunity of teleost gills. In this study, we provide the first structural and functional characterization of all teleost Igs, including secreted IgD, IgM and IgT at a mucosal surface of a teleost fish. Here we show that IgT B cells represent the major B cell subset in the gill filaments. In contrast to reported results by others, we found that all gill B cells expressing surface IgM, also expressed surface IgD and that the percentage of B cells solely expressing either IgD or IgM was negligible. We found that the majority of bacterial microbiota in the gill mucosa is coated with IgT and, to a much lesser degree with IgM and IgD. More crucially, significant specific-IgT immune responses against Ichthyophthirius multifiliis (Ich) and Flavobacterium columnare were measured in the gill mucus, while pathogen-specific IgM responses were almost exclusively detected in the serum. Pathogen-specific IgD titers were absent both in gill mucus and serum. Importantly, we found significant IgT B-cell proliferative responses in the gill but not in the spleen or head kidney of fish that survived Ich infection. Moreover we also found that Ichand F. columnare-specific IgT titers were locally produced by gill explants of survivor fish while they were absent in the spleen or head kidney explants of the same animals. In addition to showing that IgT is the main Ig player in gill mucosal immunity, the observed generation of local IgT B cell proliferative and pathogen-specific IgT responses in the gills provides the first demonstration of locally induced B cell and secretory Ig responses in the mucosa of a teleost. Moreover, this represents the first study in which a bacterial pathogen is shown to induce dominant IgT responses in a fish mucosal site, thus strongly suggesting that IgT is induced by a variety of pathogens in addition to parasites. Our findings also have special relevance from an applied perspective as they may lead to the development of fish vaccines and immunostimulants that have the capacity to induce gill IgT mucosal immune responses.

An adaptive immune response driven by mature, antigen-experienced T and B cells within the microenvironment of oral squamous cell carcinoma.

Lymphocyte infiltrates have been observed in the microenvironment of oral cancer; however, little is known about whether the immune response of the lymphocyte infiltrate affects tumor biology. For a deeper understanding of the role of the infiltrating-lymphocytes in oral squamous cell carcinoma (OSCC), we characterized the lymphocyte infiltrate repertoires and defined their features. Immunohistochemistry revealed considerable T and B cell infiltrates and lymphoid follicles with germinal center-like structures within the tumor microenvironment. Flow cytometry demonstrated that populations of antigen-experienced CD4+ and CD8+ cells were present, as well as an enrichment of regulatory T cells; and T cells expressing programmed death-1 (PD-1) and T cell Ig and mucin protein-3 (Tim-3), indicative of exhaustion, within the tumor microenvironment. Characterization of tumor-infiltrating B cells revealed clear evidence of antigen exposure, in that the cardinal features of an antigen-driven B cell response were present, including somatic mutation, clonal expansion, intraclonal variation and isotype switching. Collectively, our results point to an adaptive immune response occurring within the OSCC microenvironment, which may be sustained by the expression of specific antigens in the tumor.

Immunoreceptor TIGIT inhibits the cytotoxicity of human cytokine-induced killer cells by interacting with CD155.

T cell Ig and ITIM domain (TIGIT) is a newly identified inhibitory receptor expressed on T and natural killer (NK) cells. Cytokine-induced killer (CIK) cells express CD3 and CD56 molecules, and share functional properties with both NK and T cells. However, it remains unknown whether TIGIT is expressed in CIK cells. Here, we show that TIGIT is expressed by CIK cells and interacts with CD155. By blocking TIGIT using an anti-TIGIT functional antibody, we demonstrate that CIK cells display increased proliferation; higher cytotoxic targeting of tumor cells expressing CD155; and higher expression of interferon-γ (IFN-γ), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Furthermore, increases in IFN-γ and cytotoxicity by blockade of TIGIT were reduced by blocking DNAX accessory molecule-1 (DNAM-1) signaling, implying that TIGIT exerts immunosuppressive effects by competing with DNAM-1 for the same ligand, CD155. Our results provide evidence that blockade of TIGIT may be a novel strategy to improve the cytotoxic activity of CIK cells.

Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway.

NK cells accumulate at the maternal-fetal interface (MFI) and play essential roles in maintaining immune tolerance during pregnancy. The mechanisms that facilitate NK cells tolerance to fetal tissue are largely unknown. T cell Ig and mucin domain-containing protein 3 (Tim-3) is a newly defined molecule with essential immunological function in many physiological and pathological processes. Recent study showed that Tim-3 was involved in the regulation of immune tolerance at MFI. However, whether Tim-3 regulates NK cells cytotoxicity toward trophoblasts is unclear. Here, we showed Tim-3 was mainly expressed by decidual NK cells (dNK) and Tim-3 level in dNK was higher than peripheral NK cells (pNK). Tim-3+ dNK expressed more levels of mature markers CD94 and CD69 than Tim-3- dNK cells and blocking Tim-3 significantly inhibited dNK IFN-γ and TNF-α secretion. Furthermore, we found TGF-β1 may contribute to such up-regulation of Tim-3 in NK cells. Interestingly, blocking Tim-3 enhanced NK cytotoxicity toward trophoblast cell line HTR-8 but not K562. We found HTR-8 expressed Tim-3 ligand Galectin-9, in contrast K562 did not. Small interfering RNA-mediated silencing of Galectin-9 expression enhanced NK cytotoxicity toward HTR-8. We further showed Tim-3/Galecin-9 inhibited NK cytotoxicity toward trophoblast partially via impairing the degranulation process. In addition, clinical data showed that abnormal Tim-3 level on pNK might be associated with recurrent spontaneous abortion (RSA). Thus, our data demonstrate Tim-3/Galectin-9 pathway maintains local tolerance by suppressing NK cytotoxicity toward trophoblasts which may represent a new immunologic tolerance mechanism at MFI.

The amelioration of composite tissue allograft rejection by TIM-3-modified dendritic cell: Regulation of the balance of regulatory and effector T cells

T cell-dependent immune responses play a central role in allograft rejection. Exploring ways to disarm alloreactive T cells represents a potential strategy to promote long-term allograft acceptance and survival. T cell Ig domain and mucin domain 3 (TIM-3) has previously been demonstrated as a central regulator of T helper 1 (Th1) responses and immune tolerance. Hence, TIM-3 may be an important molecule for decreasing immunological rejection during composite tissue allotransplantation (CTA). In this study, BALB/c and C57BL/6 mice were chosen as the experimental animals. The effects of TIM-3 on allograft rejection were explored using TIM-3-modified mature dendritic cells (TIM-3 mDCs). A laser speckle blood flow (LSBF) imager was used to evaluate blood distribution of the BALB/c mice. ELISA, MTT, ELISPOT assays and flow cytometry analysis were carried out for further researches. We found that TIM-3 could obviously prolong the survival time of the transplanted limbs. And TIM-3 could mitigate the immune response and thus enhance immune tolerance after CTA. Also, TIM-3 can induce lymphocyte hyporesponsiveness, including facilitating lymphocyte apoptosis, decreasing lymphocyte proliferation, and influencing the secretion of inflammatory cytokines by CD4+ T cells. Furthermore, TIM-3 overexpression could induce CD4+ T cells to differentiate into regulatory T cells (Tregs), which recalibrate the effector and regulatory arms of the alloimmune response. In summary, we concluded that TIM-3 can mitigate allograft rejection and thus enhance immune tolerance by inducing lymphocyte hyporesponsiveness and increasing the number of Tregs of the alloimmune response. TIM-3 may be a potential therapeutic molecule for allograft rejection in CTA.

PD-1 blockade upregulate TIM-3 expression as a compensatory regulation of immune check point receptors in HNSCC TIL

Programmed Death 1 (PD-1) and T cell Ig and mucin domain-3 protein (Tim-3) are immune check point receptors that are upregulated on tumor infiltrating lymphocytes (TIL) in tumor-bearing mice and humans. As anti-PD-1 single agent response rates are still relatively low (20%) in HNC patients, it is important to learn how different inhibitory check point receptors work together to maintain the suppressive status of immune system. We observed that PD-1 and Tim-3 co-expression is associated with an exhausted phenotype of HNSCC TIL of patients, demonstrating the highest PD-1 levels in Tim-3 double positive TIL. We also observed that PD-1+/Tim-3+ TIL manifest dampened in Akt/p-S6 activation upon TCR stimulation, leading us to infer the potential for signaling cross-talk between PD-1 and Tim-3 downstream signaling pathways. Indeed, in freshly isolated HNSCC TIL, PD-1-/TIM-3+ T cells showed higher baseline expression of p-SHP-2 (p < 0.01) which is triggered upon PD-1 ligation. In addition, PD-1 blockade using nivolumab of human HNSCC TIL led to upregulation of Tim-3 expression, suggesting a circuit of compensatory, cross-talk signaling and permitting escape from anti-PD-1 blockade during TCR stimulation. In a murine HNC tumor model, anti-PD-1 treatment modestly suppressed tumor growth, but in TIL from persistent tumors in these mice, Tim-3 expression was dramatically upregulated after PD-1 blockade. Taken together, in response to PD-1 blockade, the most exhausted TIL appear to upregulate Tim-3 as a compensatory mechanism, supporting dual targeting, which may provide new therapeutic strategy for cancer immunotherapy. Figure 1 Figure 2 Figure 3 Figure 4

Deleted in Breast Cancer 1 Suppresses B Cell Activation through RelB and Is Regulated by IKKα Phosphorylation.

Alternative NF-κB signaling is crucial for B cell activation and Ig production, and it is mainly regulated by the inhibitor of κ B kinase (IKK) regulatory complex. Dysregulation of alternative NF-κB signaling in B cells could therefore lead to hyperactive B cells and Ig overproduction. In our previous, study we found that deleted in breast cancer 1 (DBC1) is a suppressor of the alternative NF-κB pathway to attenuate B cell activation. In this study, we report that loss of DBC1 results in spontaneous overproduction of Ig in mice after 10 mo of age. Using a double mutant genetic model, we confirm that DBC1 suppresses B cell activation through RelB inhibition. At the molecular level, we show that DBC1 interacts with alternative NF-κB members RelB and p52 through its leucine zipper domain. In addition, phosphorylation of DBC1 at its C terminus by IKKα facilitates its interaction with RelB and IKKα, indicating that DBC1-mediated suppression of alternative NF-κB is regulated by IKKα. Our results define the molecular mechanism of DBC1 inhibition of alternative NF-κB activation in suppressing B cell activation.

TIGIT predominantly regulates the immune response via regulatory T cells.

Coinhibitory receptors are critical for the maintenance of immune homeostasis. Upregulation of these receptors on effector T cells terminates T cell responses, while their expression on Tregs promotes their suppressor function. Understanding the function of coinhibitory receptors in effector T cells and Tregs is crucial, as therapies that target coinhibitory receptors are currently at the forefront of treatment strategies for cancer and other chronic diseases. T cell Ig and ITIM domain (TIGIT) is a recently identified coinhibitory receptor that is found on the surface of a variety of lymphoid cells, and its role in immune regulation is just beginning to be elucidated. We examined TIGIT-mediated immune regulation in different murine cancer models and determined that TIGIT marks the most dysfunctional subset of CD8+ T cells in tumor tissue as well as tumor-tissue Tregs with a highly active and suppressive phenotype. We demonstrated that TIGIT signaling in Tregs directs their phenotype and that TIGIT primarily suppresses antitumor immunity via Tregs and not CD8+ T cells. Moreover, TIGIT+ Tregs upregulated expression of the coinhibitory receptor TIM-3 in tumor tissue, and TIM-3 and TIGIT synergized to suppress antitumor immune responses. Our findings provide mechanistic insight into how TIGIT regulates immune responses in chronic disease settings.

Direct high-throughput amplification and sequencing of immunoglobulin genes from single human B cells.

Single cell Ig gene amplification and sequencing has been widely used for the molecular and functional assessment of human antibody repertoires and has led to the identification of recombinant monoclonal antibodies with therapeutic potential against diverse pathogens 1, 2, 3. Due to the high reagent and Sanger sequencing costs, these antibody‐cloning strategies are limited to the analysis of relatively small B‐cell numbers and do not allow in‐depth repertoire measurements to assess the clonality and clonal evolution of B‐cell responses. A major goal in the field has been the development of platforms that allow the high‐throughput analysis of antibody repertoires at low costs 4. Next‐generation sequencing (NGS) of Ig heavy and light chain genes facilitates the high‐throughput analysis of antibody repertoires. So far only one platform preserves natural Ig gene associations at single‐cell level through linkage of Ig heavy and light chain amplicons before sequencing 5. However, due to the short NGS read‐lengths full‐length Ig genes are not readily available for cloning and recombinant monoclonal antibody production. Here, we describe a platform for the high‐throughput analysis of human antibody repertoires at single cell level that is fully compatible with direct Ig gene cloning and expression.

Divergent Phenotypes of Human Regulatory T Cells Expressing the Receptors TIGIT and CD226.

Regulatory T cells (Tregs) play a central role in counteracting inflammation and autoimmunity. A more complete understanding of cellular heterogeneity and the potential for lineage plasticity in human Treg subsets may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4(+)FOXP3(+)Helios(+) thymic-derived Tregs and CD4(+)FOXP3(+)Helios(-) T cells, followed by comparison with CD4(+)FOXP3(-)Helios(-) T conventional cells. These analyses revealed that the coinhibitory receptor T cell Ig and ITIM domain (TIGIT) was highly expressed on thymic-derived Tregs. TIGIT and the costimulatory factor CD226 bind the common ligand CD155. Thus, we analyzed the cellular distribution and suppressive activity of isolated subsets of CD4(+)CD25(+)CD127(lo/-) T cells expressing CD226 and/or TIGIT. We observed TIGIT is highly expressed and upregulated on Tregs after activation and in vitro expansion, and is associated with lineage stability and suppressive capacity. Conversely, the CD226(+)TIGIT(-) population was associated with reduced Treg purity and suppressive capacity after expansion, along with a marked increase in IL-10 and effector cytokine production. These studies provide additional markers to delineate functionally distinct Treg subsets that may help direct cellular therapies and provide important phenotypic markers for assessing the role of Tregs in health and disease.

TIGIT facilitates liver regeneration via limiting natural killer cell over-activation (INM6P.341)

Abstract Innate immune cells, particularly natural killer (NK) cells, are known to negatively regulate liver regeneration, and inhibition of NK cell overactivation could facilitate liver regeneration. However, the molecular mechanisms that limit NK cell overactivation during liver regeneration are still elusive. In our study, we show that T cell Ig and ITIM domain (TIGIT), a coinhibitory receptor, is selectively up-regulated on NK cells, along with high expression of its ligand, poliovirus receptor (PVR/CD155), on hepatocytes during liver regeneration. Moreover, TIGIT absence impairs liver regeneration in vivo, along with NK cell overactivation and higher IFN-γ production by NK cells. We also show that NK cell-derived higher IFN-γ production is the major account of impaired liver regeneration caused by the absence of TIGIT. Adoptive transfer of Tigit-/- NK cells into NK-deficient Nfil3-/- mice sufficiently led to impairment of liver regeneration. On the other hand, silencing PVR in hepatocytes rescued impaired liver regeneration caused by TIGIT deficiency in vivo, while blockade of TIGIT in NK-hepatocyte coculture increased IFN-γ production by NK cells in vitro. Therefore, these results reveal that TIGIT is a safeguard molecule to facilitate liver regeneration through negatively regulating NK-hepatocyte crosstalk.

KIR and HLA genotypes predictive of low-affinity interactions are associated with lower relapse in autologous hematopoietic cell transplantation for acute myeloid leukemia.

Killer cell Ig-like receptors (KIRs) bind cognate HLA class I ligands with distinct affinities, affecting NK cell licensing and inhibition. We hypothesized that differences in KIR and HLA class I genotypes predictive of varying degrees of receptor-ligand binding affinities influence clinical outcomes in autologous hematopoietic cell transplantation (AHCT) for acute myeloid leukemia (AML). Using genomic DNA from a homogeneous cohort of 125 AML patients treated with AHCT, we performed KIR and HLA class I genotyping and found that patients with a compound KIR3DL1(+) and HLA-Bw4-80Thr(+), HLA-Bw4-80Ile(-) genotype, predictive of low-affinity interactions, had a low incidence of relapse, compared with patients with a KIR3DL1(+) and HLA-Bw4-80Ile(+) genotype, predictive of high-affinity interactions (hazard ratio [HR], 0.22; 95% confidence interval [CI], 0.06-0.78; p = 0.02). This effect was influenced by HLA-Bw4 copy number, such that relapse progressively increased with one copy of HLA-Bw4-80Ile (HR, 1.6; 95% CI, 0.84-3.1; p = 0.15) to two to three copies (HR, 3.0; 95% CI, 1.4-6.5; p = 0.005) and progressively decreased with one to two copies of HLA-Bw4-80Thr (p = 0.13). Among KIR3DL1(+) and HLA-Bw4-80Ile(+) patients, a predicted low-affinity KIR2DL2/3(+) and HLA-C1/C1 genotype was associated with lower relapse than a predicted high-affinity KIR2DL1(+) and HLA-C2/C2 genotype (HR, 0.25; 95% CI, 0.09-0.73; p = 0.01). Similarly, a KIR3DL1(+) and HLA-Bw4-80Thr(+), HLA-Bw4-80Ile(-) genotype, or lack of KIR3DL1(+) and HLA-Bw4-80Ile(+) genotype, rescued KIR2DL1(+) and HLA-C2/C2 patients from high relapse (p = 0.007). These findings support a role for NK cell graft-versus-leukemia activity modulated by NK cell receptor-ligand affinities in AHCT for AML.

Tim-1 is essential for induction and maintenance of IL-10 in regulatory B cells and their regulation of tissue inflammation (IRC11P.435)

Abstract T cell Ig and mucin domain (Tim)-1 identifies IL-10-producing regulatory B cells (Bregs). Mice on the C57BL/6 background harboring a loss of function Tim-1 mutant showed progressive loss of IL-10 production in B cells and with age developed severe multi-organ tissue inflammation. We demonstrate that Tim-1 expression and signaling in Bregs are required for optimal production of IL-10. B cells with Tim-1 defects have impaired IL-10 production but increased proinflammatory cytokine production including IL-1 and IL-6. Tim-1-deficient B cells promote Th1 and Th17 responses but inhibit the generation of regulatory T cells (Foxp3+ and IL-10-producing type 1 regulatory T (Tr1) cells) and enhance the severity of experimental autoimmune encephalomyelitis (EAE). Mechanistically, Tim-1 on Bregs is required for apoptotic cell (AC) binding to Bregs and for AC-induced IL-10 production in Bregs. Treatment with AC reduces EAE severity in wildtype (WT) but not Tim-1-deficient Bregs. Collectively, these findings suggest that in addition to serving as a marker for identifying IL-10-producing Bregs, Tim-1 is also critical for maintaining self-tolerance by regulating IL-10 production in Bregs.

Boosting antitumor immunity through TIM1 (IRM11P.631)

Abstract The lack of co-stimulation, as well as the dominance of co-inhibition, of type 1 lymphocytes, such as CD8+ T cells and NK cells, is a principal immunosuppressive mechanism in the tumor microenvironment. Conversion of the tumor immunosuppressive microenvironment to one that favors cancer-eradicating immune responses is therefore a goal for the development of more effective cancer immunotherapy. Targeting co-stimulatory pathways represents one attractive therapeutic strategy. T cell Ig mucin (TIM) proteins are structurally related to type I membrane glycoproteins. Previous studies have shown that TIM-1 is highly expressed on activated CD4+ T cells, facilitates the activation of Th1 cells, Th17 cells, and Th2 cells, and is implicated in the pathogenesis of human allergy and autoimmune diseases. These data support an important role for TIM-1 in regulating CD4+ T cell function. However, the role for TIM-1 in CD8+ T cell- or NK cell-mediated immune responses, including antitumor immune responses, is unknown. Here we showed that tumoral expression of antitumor cytokines such as IL-12 and IL-33 upregulated TIM-1 on tumor-infiltrating CD8+ T cells and NK cells. In addition, we demonstrated that TIM1 mAbs greatly inhibited tumor growth, resulting in greatly elevated cellular immune responses against tumor. The in-depth mechanistic insights and implication in tumor immunity are gained by our studies. Our study establishes TIM-1 as a potential target for enhancing antitumor immunity.

TIGIT and PD-1 impair tumor antigen-specific CD8⁺ T cells in melanoma patients.

T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen-specific (TA-specific) CD8⁺ T cells and CD8⁺ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8⁺ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8⁺ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1⁺TIM-3⁺ TA-specific CD8⁺ T cells. PD-1⁺TIGIT⁺, PD-1⁻TIGIT⁺, and PD-1⁺TIGIT⁻ CD8⁺ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand-expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8⁺ T cells and CD8⁺ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8⁺ T cells and CD8⁺ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8⁺ T cell responses in patients with advanced melanoma.

Stereotypic rheumatoid factors that are frequently expressed in mucosa-associated lymphoid tissue-type lymphomas are rare in the labial salivary glands of patients with Sjögren's syndrome.

Among autoimmune diseases, Sjögren's syndrome (SS) is most strongly associated with the development of malignant B cell lymphoma, in particular mucosa-associated lymphoid tissue (MALT)-type lymphoma. Previously, we have shown that in ∼40% of cases of salivary gland MALT lymphoma, high-affinity stereotypic rheumatoid factor (RF) B cell receptors, specific for IgG-Fc, are expressed. This study was undertaken to investigate whether in the inflamed salivary glands of patients with SS, a similar RF-biased Ig repertoire is present. Extensive analyses of the B cell Ig VH region repertoire were performed on microdissected tissue samples from the labial salivary glands of 4 patients with SS. All SS labial salivary glands harbored expanded B cell clones, of which 1 or 2 were highly expanded and detected in >50% of the microdissected samples. However, among the identified 464 distinct Ig clonotypes, only 3 stereotypic RF-expressing clones were detected. In 2 patients with SS, an RF-expressing clone was detected at low frequency in 1 of the microdissected samples, whereas 1 patient with SS harbored a highly expanded RF-expressing clone that was detected in all microdissected samples and also detected in the peripheral blood. Two years after analysis of this sample, the latter patient developed a diffuse large B cell lymphoma originating from the same RF clone. Inflamed labial salivary glands in patients with SS generally harbor 1 or 2 highly expanded B cell clones. The repertoire strongly biased toward stereotypic RFs in salivary gland MALT lymphomas is not a reflection of a similar repertoire in the inflamed salivary glands of patients with SS; rather, in the latter, the repertoire is based on a strong selection advantage of incidental stereotypic RF-expressing B cells.